Trying to Crack An Icy Mystery: Cryogenetic Secrets May Aid Organ Transplants
By David A. Fahrenthold
Washington Post Staff Writer
Sunday, December 12, 2004; Page C01

This is the way a wood frog freezes:

First, as the temperature drops below 32 degrees, ice crystals start to form just beneath the frog's skin. The normally pliant and slimy amphibian becomes -- for lack of a better word -- slushy.

Then, if the mercury continues to fall, ice races inward through the frog's arteries and veins. Its heart and brain stop working, and its eyes freeze to a ghostly white.

"Imagine an ice cube. Paint it green," and you've got the wood frog in winter, said Ken Storey, a professor at Carleton University in Ontario. The frog is solid to the touch and makes a mini-thud when dropped.

But it is not dead. When a thaw comes, the frog is able to melt back into its normal state over a period of several hours, restart its heart and hop away, unscathed.

This amazing process of reanimation -- repeated every winter in the woods of Maryland, Virginia and the District -- is being examined by scientists hoping to learn the secrets of the frog and other animals that freeze solid.

The hope is that these apparent Lazarus routines can yield clues for improving human medicine, including better preservation of organs on their way to transplant patients.

"Here's an amphibian that has solved the problem of cryo-preserving its organs -- all of them, simultaneously," said Jon Costanzo, a professor of zoology at Miami University in Ohio. "And we haven't been able to do that with one [human organ]."

The Washington region is actually home to several species of what scientists call "freeze-tolerant" animals. One is the wood frog, a two-inch-long creature with a call like a quack, which lives in woods from Georgia to Alaska.

Other local species -- spring peeper and the gray tree frog, as well as a few kinds of caterpillars and the babies of the painted turtle -- can freeze but lose the ability as they age.

Scientists say these animals' freezing abilities are just extreme reactions to a problem that all mid-Atlantic animals face: periodic blasts of winter cold. Human retirees head to Florida, Chesapeake Bay crabs bury themselves in the mud and most frog species hide out deep underground or underwater.

But not the freezing frogs. Instead, buried just a few inches under dirt and leaves, they welcome the chill. When the soil starts freezing -- even if it falls just a couple of degrees below 32 -- so do the frogs.

The result is something like the frozen gray tree frog that Professor Jack R. Layne Jr. held in his hand this week in a lab at Slippery Rock University in Pennsylvania.

Instead of its normal grayish-green, the frog had turned almost purple, its limbs and head stuck in contortions. It looked for all the world like a practical joke: an ice cube made to resemble a frog.

"You can see that it's quite solidly frozen," Layne said. "They kind of turn bluish."

The frogs can survive this process, in which as much as 65 percent of their body water freezes, because their cells are protected by a kind of natural antifreeze.

Scientists say that, before winter comes, the frogs eat ravenously, storing a starch in their livers. A freeze triggers their bodies to convert the starch into other compounds, most often glucose, or blood sugar. The frogs become, in essence, extremely diabetic.

The glucose lowers the freezing temperature of water inside the frogs' cells, and because of this, the cells stay liquid, even as ice fills the space around them. This is crucial: If the water inside the cells froze, scientists say, the jagged ice crystals would destroy everything inside, killing the frog.

It's very hard to find frogs frozen like this in the wild, because they're hidden underground. At the Patuxent Research Refuge, a 12,750-acre forest near Laurel, wildlife biologist Robin E. Jung of the U.S. Geological Survey, said she occasionally gets lucky and finds wood frogs hunkered down for winter.

"Just like" -- she stiffened like she'd been shot with a super-villain's ice ray -- "freezing."

In this area, cold snaps usually aren't long enough to keep the frogs frozen for more than a few days. But wood frogs live as far north as Canada and Alaska, and in those places they can freeze for months, scientists said.

Medical researchers say they hope to copy these long-term freezing abilities to add hours or even days to the time that human organs can be preserved.

Now, after organs are removed from a donor, they are packed in a special solution and kept on ice. But they can't be frozen because of the damage that ice crystals would do to the cells. Without freezing, the shelf life of these organs can be as much as 48 hours for a kidney and as little as four hours for a heart.

If organs could be preserved longer, it would allow more time for locating an organ recipient and setting up the transplant operation, said Jimmy A. Light, head of transplantation at Washington Hospital Center.

"It would allow you to have a more prepared patient," Light said. "Now, it's kind of like a fire drill. The bell rings, the clock ticks and you've got to get going."

In one experiment, University of California professor Boris Rubinsky removed a rat's liver and filled it with glycerol, hoping the chemical would act as glucose does in wood frogs.

The experiment worked: The liver was frozen, then thawed and transplanted successfully into another rat, Rubinsky said.

Other researchers have turned to arctic fish, which manufacture special chemicals to keep from freezing even as the water around them falls below 32 degrees.

Using fish proteins made in a lab, scientists have managed to preserve a pig's heart at subfreezing temperatures for 24 hours, then transplant it into another pig.

Scientists say they don't see any immediate potential for putting an entire human body in a science fiction-style deep freeze; the frogs, after all, don't stay frozen forever.

But just freezing and thawing one human organ would be a major breakthrough.

"If we can translate that into a human heart, then we'll do very well," Rubinsky said.

Now, even as researchers try to copy the frog's techniques, the freezing amphibians still haven't given up all their secrets.

Their ability to thaw puzzles scientists, who are trying to crack the process and pinpoint the trigger that restarts the frog's heart.

Whatever it is, Storey said, "it's not magic. It's physical chemistry."

Video of a wood frog thawing out from a frozen state is available on The Washington Post Web site at www.washingtonpost.com/wp-srv/mmedia/nation/120904-12v.htm.